Principles of Fluorescence Spectroscopy

40 INSTRUMENTATION FOR FLUORESCENCE SPECTROSCOPY
Figure 2.20. Laser line filter to transmit only the laser wavelength
(top) and a notch filter to reject the laser wavelength. From [15].
2.4.3. Filter Combinations
While one can obtain almost any desired filter with modern
coating technology, the design of custom filters for each
experiment is usually not practical. If a single filter is not
Figure 2.21. Interference filter to transmit selected wavelengths.
From [15].
Figure 2.22. Transmission profile of a combination of Corning and
Schott filters used to isolate protein fluorescence (top) and Indo-1 fluorescence
(bottom). Lower panel from [17].
adequate for a given experiment, it is often possible to combine
two or more bandpass filters to obtain the desired spectral
properties. This possibility is shown in Figure 2.22. The
UG-11 and WG-320 filters are often used in our laboratory
to isolate protein fluorescence. 16 For probes emitting near
450 nm, we often use a combination of Corning 4-96 and 3-
72 filters. 17 In this example the filter was selected to reject
702 nm, which was the excitation wavelength for two-photon
excitation of Indo-1. 17 This example illustrates an
important aspect in selecting filters. Filters should be selected
not only for their ability to transmit the desired wavelength,
but perhaps more importantly for their ability to
reject possible interfering wavelengths.
2.4.4. Neutral-Density Filters
Neutral-density filters are used to attenuate light equally at
all wavelengths. They are typically composed of sheets of
glass or quartz coated with a metal to obtain the desired
optical density. Quartz transmits in the UV and is preferred
unless no experiments will be done using wavelengths
below 360 nm. Neutral-density filters are described by their
optical density, and can typically be obtained in increments
of 0.1, up to optical densities of 4. It is often necessary to
adjust or match the intensity of two signals, which is conveniently
accomplished using neutral-density filters.